The notion of gene therapy to insert novel genes or corrected genes into cells of humans as a form of medical therapy has been a dream since at least the 70's. This dream was spurred on by the many advances made in molecular biology, with the ability to analyze and change segments of DNA. One such advance arguably has to include the technique of polymerase chain reaction (PCR). This technique involves repeated amplification cycles of copying the sequence identified by primer sets, each new round beginning with the dissociation of the newly transcribed double stranded cDNA, reannealing of primers, and ‘primer extension’. Once this was adapted to gene sequencing, it reduced the amount of time to sequence several hundred based pairs from about a week to a matter of hours or minutes. And so the Human Genome project was completed many years ahead of predictions.
With the completion of the sequencing of the genome, it was discovered the human genome contains many forms of repetitive elements one of these being endogenous retroviruses, or remnants of endogenous retroviruses. By far, most of these are not much more than a set of no longer related left and right long terminal repeats (LTRs). Perhaps as much as 5 to 8% of the human genome contains bits and pieces of endogenous retroviruses. The more complete forms are fewer in number and constitute about 0.2% of the human genome. When one considers how big the genome is (3.4 trillion base pairs), that is a lot of DNA taken up by invading “retroelements’.
This saturation of the genome by once mobile elements and the passing on from generation to generation, has caused many to wonder what are they doing there. There is growing evidence that these endogenous retroviruses may play important biological roles. These roles include the formation of syncytiotrophoblast in the development of the human placenta. For human endogenous retroviruses of the L type (HERV-L), interference with exogenous viral replication through expression of antisense mRNA, is another proposed role. Many postulate these once mobile elements may have contributed to genomic diversity and thus, evolution of the species. The expression of endogenous retroviruses (and partial forms) has been linked to disease, particularly chronic diseases, and is more frequent with aging. Many of these illnesses may be characterized by autoimmune activity (diabetes, multiple sclerosis, arthritis etc.) and in others, neurodegeneration (Alzheimer's, Parkinson's, and dementia associated with aging).
To date no gene therapy has received marketing approval in Canada or in the United States despite the fact the first gene therapy was performed on Sep. 14, 1990. There are many problems, not the least of which concerns immunogenicity issues. This term refers to the notion that vectors used for gene transfer are foreign to humans and this enables humans to mount immunological responses both antibody based and cell mediated. This means after the first exposure there is a risk of an immunological reaction with each subsequent injection. Sometimes these reactions are manageable, other times they are not and can be deadly. Gene therapy clinical trials were halted in 1998 in the US upon death of a young male adult following an anaphylactic reaction to an adenovirus vector.
Other untoward side effects of gene therapy even when performed ex vivo (cells are transfected in the laboratory and then re-injected back to the same individual) concerns leukemia. In 2002, a first then second case of leukemia occurred in clinical trials in France using the murine leukemia virus as a vector, and gene therapy clinical trials were halted. Here the retrovirus vector, genetically devoid of transforming sequences, nevertheless led to cancer due to insertional mutation (first case) and insertional activation (second case) of a normal gene LMO-2, an oncogene responsible for childhood leukemias. The safety and efficacy of gene therapies is yet to be shown particularly for retroviral vectors derived from retroviruses that are known to naturally induce leukemias.
Additional protective strategies are usually employed in the construction of gene therapy vectors to ensure only one round of replication occurs. This is because to date, the vectors chosen have been derived from disease associated animal viruses and one would not want to start a new epidemic if fully functional infectious vectors were instead used. They are usually built in two or more pieces, so that the functional genes required for packaging the cDNA are provided on a separate element from those genes needed for integration into the host genome. Because these various parts are on different strips of cDNA, this only permits one round of replication and one chance for integration if derived from the “packaging cell” where both elements have been transfected.
Restricting replication to a single round helps to prevent the establishment of a viremia (which thereby decreases the chance of adverse effects such as a leukemia for a leukemic retrovirus, or immune deficiency for an AIDS like lentivirus). However, this has the disadvantage that most host cells will not be transfected by the vector and thus the gene is not delivered to sufficient number of cells in the host for the therapy to have value. This intrinsic limitation of retroviral vectors injected in vivo, is why for blood related disorders, usually bone marrow stem cells are isolated, transfected in vitro, and tested and enumerated before being re-implanted back into the host.
Human endogenous retroviruses (HERVs) constitute about 0.5% of the human genome, but the only HERV family known to express virus-like particles is HERV-K (1-3). None of the HERVs described so far has been shown to be infectious (3), but genetic evidence suggests some members of HERV-K, such as HERV-K102, K107-K109, K113 13, might be either infectious or at least recently active in reintegrating within the genome (4). Up to 50 different copies of HERV-K are present in the human genome, but few of these contain full-length genes encoding viral structural proteins (reviewed in 1). According to a more recent paper, 41/59 of long viral open reading frames (vORFs) are HERV-K betaretroviral and the human genome contains “intact” 17 gag, 13 pol and 29 envelope genes. Five of 29 envelope genes carry a specific 292 by deletion and are Type I HERV-K (human mouse mammary tumor virus-like group 2 (HML-2)) (Villensen et al., 2004).
The prototype HERV-K10 was first identified in the human genome by virtue of its homology to the exogenous mouse mammary tumor virus (MMTV) (5), although HERV-K10 is thought to be defective (5). Subsequently 6 groups with homology to the mouse mammary tumor virus were identified and were named HML-1 through HML-6 (where HML refers to Human Mammary tumor virus Like) (6, 7). More recently 25 HERV-K10-like elements related to HERV-K102 (belonging to the HML-2 subfamily) have been described (7). Many HERV-K proviruses have been mapped and cloned (8,9) through the human genome project. These analyses have further revealed there are two types of HERV-K (HML-2) proviral genomes differing by the presence (Type II) or absence (Type I) of a 292 bp segment at the pol-env boundary (10). HERV-K102 (GenBank AF164610), a member of the Type I family, has been mapped to chromosome 1 and is closely related to K10 (M14123, Type I), K101 (AF164609 Type I), K103 (AF164611, Type I) K107 (AF164613, Type I), K108 (AF164614, AF074086, Type II at 7p22.1), K109 AF164615 Type II, and as well as K113 (GenBank AY037928, Type II) at about 98% homology at the nucleotide level when one excludes the gap at the pol-env boundary in Type I (10, 11). For the remainder of this disclosure, HERV-BZU will refer to both Type I and Type II HERV-K (HML-2) as they are related at about 98% homology.
No HERV has been shown to be infective nor has an infectious foamy retrovirus (spumavirus) originating from humans yet been found. (Heinkelein et al., 2005).
Since Type I HERV-K (HML-2) particles are expressed in the placenta (Simpson et al., 1996), and become reactivated in the adult under certain conditions, most likely this means humans would be immunologically tolerant to these particles and associated proteins. Thus, the vector would be less likely to cause disease. This would provide a distinct advantage over current gene therapy vectors as there would be little risk of immunological or other adverse reactions using HERV-BZU as the vector. Thus, HERV-BZU could be repeatedly injected for one purpose, or could be subsequently used for a different purpose with minimal risk of anaphylactic shock or other immunological adverse reactions. Additionally, HERV-BZU lacks the immunosuppressive domain located within the transmembrane region of most retrovirual envelope antigens, and thus, this again reduces the risk of pathological events.
Current retroviral vectors such as Murine Leukemia Virus (MLV) vectors have additional limitations in that cells must be replicating in order for infection and integration to occur. It is possible that a HERV-BZU type vector in analogy to foamy virus vectors, may infect both non-replicating and replicating cells, indicating a broader usefulness to target many cell types. In this regard it would be particularly suited to stem cell gene therapy, as many stem cells exist in non-replicative phases. It is recognized that the combination of gene therapy with autologous stem cell therapy is one area of medicine expected to grow significantly over the next few years. This is expected to have the most potential for more immediate clinical applications as the transfection occurs in vitro, is more easily controlled, and can be tested for any unexpected alterations before injection back into the host.
Overall foamy virus or foamy-virus-like vectors have many advantages over other retroviral vectors (Trobridge et al, 2002, Hill et al, 1999, Mergia & Heinkelein, 2003). Indeed when replication competent, they can and do infect many cell types in the body, and can cross the blood brain barrier (important for therapy of neurological disorders) (Heinkelein et al, 2005. They do not and have not been associated with disease whether naturally or experimentally transmitted. But there are three main concerns about using replication competent virus vectors: 1) integration could lead to insertional mutagenesis, 2) integration could lead to activation of oncogenes and thus tumor formation, 3) recombination could lead to a new epidemic, for example like HIV. The integration sites of foamy viruses starts with “TGTG” and is evidence of an asymmetric integration process. This type of integration appears to be common to HERV-BZU, as all family members also contain the “TGTG” sequence motif at the 5 end of the provirus but not other HERVs (except HERV-L which lacks env and thus cannot be active). According to a recent investigation, the preferred integration site for foamy viruses appear to be CpG islands rather than expressed genes, making foamy virus vectors the least likely to be associated with insertional mutations of genes (Trobridge et al, 2006). As well, foamy virus was recently found to be naturally oncolytic (Heinkelein et al, 2005), a unique feature which would safeguard the host from inadvertent tumor formation. Apparently foamy viruses cannot be pseudotyped by Env from other types of retroviruses (Meirering & Linial, 2001) making recombinants with exogenous retroviruses significantly more difficult. Along this same line Patience et al, 1998 have already shown Type I HERV-K particles do not package sequences from exogenous retroviruses, indicating HERV-BZU based vectors would be less likely than most to generate recombinants.
Finally, HERV-K102 based foamy like viral vectors, are not expected to increase the risks associated with its use over and above normal, as this virus naturally replicates in humans (see below) and has existed in the human genome since the divergence of man from apes, some 5 million years ago. To the best of our knowledge, there is no other existing or proposed virus gene therapy vector which has this proven track record. As also elucidated below, we provide evidence that HERV-K102 expression is directly antagonistic to HIV infectivity (see model and explanation), and we additionally propose may be a newly described host defense system against viral and tumor transformation. Thus, we suggest if gene therapy is to move forward for the cure of various diseases or for the prevention and control of intractable infectious diseases (like HIV and prion diseases), we predict only HERV-BZU will be able to safety and effectively transfect genes or gene products.